Protein C, a naturally occurring plasma protein, is a serine protease precursor that can be converted to the active enzyme, activated protein C (APC). Current knowledge supports the broad concept that the protein C system provides tissue homeostasis when many types of cells and organs are subjected to various injuries. APC exerts two major, distinct activities: (1) anticoagulant activity and (2) cytoprotective direct effects on cells. APC's anticoagulant activity targets factors Va and VIIIa in reactions requiring the cofactor, plasma protein S. Based on APC-initiated cell signaling, the cytoprotective activities of APC potentially comprise anti-apoptotic and anti-inflammatory activities, alterations of gene expression, and stabilization of endothelial and epithelial barriers. For cell signaling initiation activation of protease activated receptor1 (PAR1) by APC bound to another receptor (endothelial protein C receptor) is a key paradigm. Other receptors play key roles, and there is a major gap in basic knowledge about APC receptors and mechanisms that directly bind APC. This project is focused on providing key basic knowledge about APC's interactions with three of its key receptors, PAR1, Mac-1 (M2, CD11b/CD18, C3 receptor), and apoER2 (LRP8) and with its anticoagulant cofactor, protein S. The project is based on several hypotheses, each of which is strongly supported by preliminary data. We hypothesize that PAR1 cleavage at Arg46 mediates APC-initiated biased, cytoprotective signaling. Using the latest methods for obtaining GPCR x-ray structures, we expect to obtain 3-dimensional x-ray crystallographic structures of at least three different conformations of PAR1 to define the basis for biased signaling. The N- terminal tail of PAR1 provides tethered ligands are agonists for biased signaling. We will use synthetic peptides combined with studies of PAR1 mutants to decipher the tale of the various tails of PAR1 that can cause intra- molecular agonism. We expect to obtain insights into where and how the novel PAR1 Asn47-tail, compared to the Ser-42 tail, binds to PAR1. We hypothesize that the alpha-M I-domain of Mac-1 and multiple domains of apoER2 can bind APC on partially overlapping, extended surfaces of APC. Strong preliminary data delineated the areas on APC that merit interrogation. Using mutagenesis and appropriate binding protocols, we will identify the surfaces on APC that bind each receptor. Using mutagenesis of receptors, we will identify residues and areas on each receptor that bind APC. Based on preliminary studies of novel APC mutants, we hypothesize that protein S binds to an extended APC surface involving multiple residues that span an area from the top of the Gla domain over the EGF1 domain and onto the top of the EGF2 domain. We expect to map completely the protein S binding surface on APC. New knowledge about APC-receptor interactions could give rise to novel biologics involving 2nd or 3rd generation APC mutants with beneficially modified specificities.